Work will be carried out on three interrelated projects. One of these will involve acetyl-CoA carboxylase, another beta-hydroxy-beta-methylglutaryl coenzyme A reductase, and the third cholesterol 7 alpha-hydroxylase. Studies with the first two of these enzymes will be concerned with the regulation of their activities, both short-term and long-term. The short-term regulation involves phosphorylation-dephosphorylation of enzyme and the long-term regulation is effected by a change in the quantity of enzyme. Our studies will be concerned with the isolation and purification of the phosphorylating-dephosphorylating enzymes and the mechanisms of regulation of these enzyme activities. Our long-term regulation studies will be concerned with the isolation, purification and translation of mRNA for each of the enzymes and the mechanism of regulation of the of the synthesis of mRNA for each. Of particular interest is the mechanism of hormonal regulation of these enzymes. Studies with cholesterol 7 alpha-hydroxylase will first attempt to purify this enzyme to homogeneity. When this is accomplished we will investigate the mechanism of regulation of activity of this enzyme. Studies on the regulation of enzyme activity will involve the use of intact animals, isolated cells and tissue culture cells. Hormonal effects have been, or will be, demonstrated with each of these systems. Studies on the isolation of mRNA will involve the use of 125I-labeled monospecific antibody to precipitate polysomes containing a particular mRNA. Assays will be carried out to determine the quantity of immunoprecipitable polysomes as a function of the nutritional or hormonal state of an animal. Subsequently, mRNA will be isolated, purified and translated in either a wheat germ or reticulocyte lysate protein synthesizing system. Precedent for the latter studies will be the studies that we have already carried out on the mRNA for the rat liver fatty acid synthetase complex.